Antisense oligonucleotide (AON) can potentially inhibit the protein synthesis by translation arrest/steric blocking or by RNase H mediated degradation of the AON/RNA hybrid. Other methods of gene silencing include formation of triplexes by base-pairing with double-helical DNA (antigene effects), or RNA interference (RNAi), by a short double-stranded RNA (siRNA). The in vivo application of the gene silencing technology warrants chemical modification of the antisense/antigene or siRNA strand to enhance the target affinity, specificity, stability in the blood serum and tissue specific delivery in order to improve overall pharmacokinetic properties. Various modifications of oligonucleotide involving sugar, phosphodiester linkage and nucleobase are known. Of these the phosphorothioate7 backbone modified oligonucleotides have found some use in therapeutics. Recent years have seen development of conformationally-constrained bicyclic (FIG. 1) and tricyclic nucleotides, in which the sugar is locked in a definite puckered conformation. Such oligonucleotides show promising properties with respect to the target RNA binding and nuclease resistance. Among several molecules reported, short nucleotides containing LNA1 (≡BNA2) modifications have shown unprecedented thermal stability (+3 to +8° C. per modification depending upon the sequence context). The enhanced target binding property of the North-conformationally constrained bicyclic sugar units in these nucleotides has been attributed to the improved stacking between the nearest neighbors and quenching of concerted local backbone motions by LNA nucleotides in ssLNA so as to reduce the entropic penalty in the free energy of stabilization for the duplex formation with RNA. These bicyclic constrained analogs have thus been extensively used to facilitate the down-regulation of genes. The features of LNA/BNA has led to the synthesis of a number of closely related analogs, in which the 2′,4′-bridge has been altered3 or a new type of 1′,2′-bridged constraint has been introduced, such as in 1′,2′-oxetane4 or 1′,2′-azetidine5 analog. Such modifications show similar or moderately depressed Tm properties when compared to LNA, but the nuclease resistance or RNase H recruitment properties (for example, ENA,6 PrNA,7 and aza-ENA8) have turned out to be relatively more favorable than those exhibited by the LNA-containing AONs.
Studies with modified nucleotides show that substituents play important role in conformational steering, controlling hydration, inducing hydrophobic/hydrophilic interactions, and generally using the electrostatic interactions to neutralize, for example, the phosphates charge, as well as to influence interaction of modified oligonucleotide with other nucleotides and/or enzymes present in the system. We and others have argued that replacement of the hydrophilic 2′-oxygen of LNA or ENA or 2′-nitrogen from their amino analogs by the hydrophobic carbocyclic analogs would steer both target affinity as well as the nuclease stability in the blood serum because of change in the immediate shell of hydration. Recently,14 the ring-closing metathesis approach had been employed to synthesize two carbocyclic analogs of ENA with three carbons locking the C2′ and C4′ (compounds K/L in FIG. 1). These carbocyclic analogs had been incorporated into AONs with natural phosphodiester backbone which lead to increased thermal stability (Tm) by 2.5-4.5° C./modification with the complementary RNA. However, no blood serum or 3′-exonuclease stability or the RNase H recruitment capability of these carbocyclic analogs has so far been reported.